Numerous studies have been published which point out the strong correlation which exists between levels of high density lipoprotein (HDL) cholesterol and reduced risk of clinically evident atherosclerosis. Thus the fractionation of cholesterol and the measurement of isolated fractions is of great diagnostic import.
Typically it is useful to separate out the high density lipoproteins from other serum components, most specifically from the low and very low density lipoproteins (LDL and VLDL), and then measure the HDL cholesterol by some form of assay, such as some condensation reaction, or enzyme assay. One method to effect such a separation utilizes the interaction between LDL and VLDL and polyanions and (Group II) metal ions, as exemplified by heparin and Ca. One such method known in the art is the heparin/MnCl.sub.2 precipitation approach which utilizes heparin and MnCl.sub.2 to precipitate out LDL and VLDL leaving the HDL in the serum solution. The precipitation approach for fractionation of cholesterol is described in Clin. Chem. 22/11, 1812-1816 (1976) and Clin. Chem. 18/6, 499-502 (1972).
In the practical application of these fractionation or separation procedures only minute quantities of the active ingredients, i.e., heparin and the divalent cation salt need be utilized. Accordingly, it is highly desirable to be able to premeasure these small quantities of active ingredients and to put them in a useful form in order to give precision and standardization to the assay procedure. Unfortunately, the required quantities are so small as to be virtually invisible to the naked eye. Thus, in addition to the difficulties inherent in measuring such small amounts of these key active ingredients, it is exceedingly difficult to move or handle the premeasured amounts without danger of some loss. For example, attempts to provide heparin and MnCl.sub.2, premeasured and lyophilized, for use in the HDL cholesterol assay have produced unacceptable non-uniform results.
These disadvantages are overcome when the active ingredients for the separation of LDL and VLDL from HDL cholesterol are dispersed in an inert filler comprising a polysaccharide, a terminal interlocking linear glucose polymer and a vinylpyrrolidone. The filler not only adds bulk to the reagent mixture but does so without influencing the interaction of the active ingredients with the low and very low density lipoproteins. The presence of the filler prevents loss of heparin and divalent cation salt during lyophilization and/or other handling during which the active ingredients are put in forms useful for standardized testing, such as tablets.
Moreover, it has been found that when the reagent mixture of the present invention is employed in a HDL separation, the cholesterol containing serum sample itself acts as both the solvent for the reagent composition and as the milieu of the reaction.